Skin aging is a complex process that involves metabolic and physiologic changes that lead to an increasing susceptibility to disease and ultimately death. Besides chronological aging and its impact on human skin and its physiological processes and functions, a number of environmental factors and exposures also have a marked impact upon skin aging, or at least the appearance and manifestation of those consequences associated with or arising from skin aging. Perhaps no environmental factor or exposure is more detrimental to the skin than the exposure of skin to UV radiation which causes a number of diverse biological effects, including sunburn (inflammation), induction of skin cancer (melanoma), premature skin aging, and alteration in cutaneous immune cells (immunosuppression), all of which lead to damage, including permanent damage, of the skin cells. Unfortunately, skin cell damage due to UV radiation is induced by several mechanisms such as UV-induced immuno-suppression, UV-induced DNA damage and accumulation of DNA damaged products, such that efforts to protect the skin, for example, by application of sunscreen, application of moisturizers, post-sunburn treatments and the like, while effective in some respects, are ineffective in others. Rather, for comprehensive photo-protection, especially against premature skin aging, photo-allergies, immune-suppression and skin cancer, it is believed necessary to reverse or reduce UV-induced biochemical changes in the skin.
However, in order to undertake efforts to reverse or reduce UV-induced biochemical changes in the skin one must first appreciate and understand the various UV-induced changes and damage that must be addressed and how those biochemical changes affect the skin and its physiological processes.
DNA Damage and CPDs
The DNA of young people is regulated to express the right genes at the right time to allow the human system to properly function and to protect the body from disease and attack. However, as the years pass the regulation of the DNA gradually gets disrupted: a factor that manifests itself in aging whereby dysregulated DNA increases the risk of different forms of cancer and other diseases. Specifically, the protection of “youthful” DNA diminished. Obviously, health depends on much more than just the regulation of our DNA; however it is clear that dysregulation of the DNA is a fundamental process which increases the risk of different diseases (R C Slieker et al., Age-related accrual of methylomic variability is linked to fundamental aging mechanisms, Genome Biology, 2016; 17 (1) DOI: 10.1186/s13059-016-10536).
There are many molecules in the skin that absorb ultraviolet (UV) radiation; however, one in particular, cellular DNA, strongly absorbs UV radiation, especially shorter wavelength solar UV radiation. It is a well-known fact that chronic exposure to UV radiation, as well as ionizing radiation, leads to DNA damage. This process underlines photo-aging, a term that broadly encompasses changes in the skin associated with life-long exposure to the sun: wrinkling, skin laxity, erythema and hyperpigmentation, among others. More importantly, from a clinical perspective, the role of DNA damage as a, if not the, key provoking event in mutagenesis and tumor development is well-documented. Indeed, DNA damage induced by ultraviolet radiation (UVR) is considered to play a direct part in the initiation, of skin cancers.
While there are various types of UV radiation induced DNA damage in the skin, the most prominent are the dipyrimidine lesions, most especially the cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidone photoproducts. CPDs play major role in skin cancer mutations relative to that of the 6-4 pyrimidine-pyrimidone photoproducts and oxidative DNA damage. Collectively, the data implicate the CPDs as the DNA lesion most strongly involved in human cancers induced by sunlight [G P Pfeifer and A Besaratinia, UV wavelength-dependent DNA damage and human non-melanoma and melanoma skin cancer, Photochem Photobiol Sci, 11:90-97, 2012]. Formation of CPDs also is a molecular trigger for solar-simulated ultraviolet radiation-induced suppression of memory immunity in humans, The mechanism is still under investigation; however, it has been found that CPDs trigger the loss of dendritic cells and infiltration by macrophages [J M Kuchel et al., Cyclobutane pyrimidine dimer formation is a molecular trigger for solar-simulated ultraviolet radiation-induced suppression of memory immunity in humans Photochem Photobiol Sci, 4(8):577-582, 2005]. All told, the CPDs constitute approximately 80% of the total lesions induced or formed by UV radiation exposure, and are believed to influence a large number of cellular functions such as replication, transcription, and DNA repair.
Recent studies have pointed to UVA as a key inducer of CPDs, but not the 6-4 photoproducts (A Tiwari et al., UVA1 induces cyclobutane pyrimidine dimers but not 6-4 photoproducts in human skin in-vivo, J Invest Dermatol, 132:394-400, 2012); whereas, UVB is found to induced both CPDs and the 6-4 photoproducts. Interestingly, the level of UVA-induced CPDs increased with epidermal depth whereas a decrease of UVA-induced CPDs was observed with UVB, suggesting that UVA may be more carcinogenic than has previously been thought.
Additionally, Brash et al have shown that chemiexcitation of melanin derivatives induces DNA photoproducts long after UV exposure has ended [S Premi, D Brash et al, Chemiexcitation of melanin derivatives induces DNA photoproducts long after UV exposure, Science, 347(6224):842-847, 2015]. These authors have further demonstrated that the presence of melanin, activation of NOS (inducible nitric oxide synthase) and NOX (NADPH oxidase), and the triplet state were required for dark CPDs formation. Hence, while one might believe the damage stops once the UV exposure is stopped, these findings indicate otherwise.
Although many, if not most, of the UV radiation induced lesions are efficiently repaired in the skin, such endogenous repair mechanisms to remove DNA lesions and damaged bases are not 100% efficient. Accordingly, CPDs formation still results in various acute effects (erythema, inflammatory responses), transient effects (suppression of immune function), and chronic effects (mutation induction and skin cancer). Despite efforts to raise awareness and teach preventative measures such as sun avoidance, the application of full-spectrum sunscreens, and the use of antioxidant creams, the incidences of both melanoma and non-melanoma skin cancers continue to increase annually and are estimated to be comparable to the sum of all other cancers combined. Given these statistics, it is clear that current preventative measures against skin cancer are insufficient. In fact, neither sunscreens nor topical antioxidants have been shown to effectively block the effects of UV radiation. Essentially, the antioxidants are of limited efficacy and, it seems, the level of these antioxidants contained in the majority of skin creams is too low to have a major impact on free radical damage. Similarly, sunscreens have limited effect as well. Sunscreens absorb only a portion of UV radiation and many fail to be photostable, oftentimes breaking down or degrading even after just a few minutes of sun exposure [H Gonzalez, N Tarras-Wahlberg, B Stromdahl, A Juzeniene, J Moan, O Larko, A Rosen, A M Wennberg, Photostability of commercial sunscreens upon sun exposure and irradiation by ultraviolet lamps, BMC Dermatology, 7:1 (2007) www.biomedicalcentral.com/1471-5945/7/1]. Furthermore, observational studies have repeatedly found sunscreen use to be associated with higher risk of cutaneous melanoma and basal cell skin cancer. This correlation is hypothesized to exist because sunscreens delay the appearance of sunburn, encouraging prolonged sun exposure and thereby increasing skin cancer risk [Yasmeen Kabir, Rachel Seidel, Braden McKnight, Ronald Moy, DNA Repair Enzymes: An Important Role in Skin Cancer Prevention and Reversal of Photodamage—A Review of the Literature, J Drugs Dermatol. 14(3):297-301, 2015].
Nitric Oxide (NO) and iNOS Activation
Nitric oxide (NO) plays a pivotal role in human physiology and pathophysiology (Oplender and Suschek, The Role of Photolabile Dermal Nitric Oxide Derivates in Ultraviolet Radiation (UVR)-Induced Cell Death, Int J Mol 14(1):191-204, 2013). It is the smallest known bioactive product of mammalian cells, is highly diffusible and reactive, and can be produced by most cell types. In the human body, NO is formed endogenously by three NO synthase enzymes. The keratinocytes express the neuronal isoform of NO synthase (nNOS), whereas the fibroblasts and other cell types in the skin express the endothelial isoform (eNOS). The third NO synthase enzyme, the inducible isoform of NO synthase (iNOS), is not expressed usually in the skin; but, under certain conditions, virtually all skin cells are capable of expressing iNOS. For example, irradiation of the skin by UVB and/or UVA radiation induces the release of inflammation transmitters, like IL-1, IL-10, TGF-β1, and TNF-α which induce iNOS to produce higher NO-concentration. iNOS can also be induced by UVB, in the absence of proinflammatory cytokines (Suschek et al., Ultraviolet A1 radiation induces nitric oxide synthase-2 expression in human skin endothelial cells in the absence of proinflammatory cytokines, J Invest Dermatol, 117:1200-1205, 2001). In following, recent studies have confirmed the role of nitric oxide (NO) as a contributor to the UV erythema response [Rhodes L E, Belgi G, Parslew R, et al. Ultraviolet-B-induced erythema is mediated by nitric oxide and prostaglandin E2 in combination. J Invest Dermatol, 117(4):880-885, 2001]. Large amounts of nitric oxide (NO) production following induction of the inducible NO synthase (iNOS) gene has also been implicated in the pathogenesis of various inflammatory diseases. Accordingly, it is generally accepted that high levels of NO are often correlated with inflammatory skin conditions as well as erythema, edema and stimulation of melanogenesis.
It has also been established that NO produced in the skin by NO synthase can combine with superoxide to form peroxynitrile, a highly reactive oxidant and mediator of tissue injury which is found to impair lipid peroxidation itself and oxidize lipid soluble antioxidants (Hogg and Kalyanaraman, Nitric oxide and lipid peroxidation, Biochim Biophys Acta, 1411:378-384, 1999). Indeed, these peroxynitrite radicals are found to react directly with several critical cellular targets including thiols, proteins, lipids and DNA (Current Pharmaceutical Design, 17(35):3905-3932, 2011). The rates of peroxynitrite production in-vivo in specific compartments have been estimated to be as high, as 50-100 μM per minute. In light of the multiple target molecule reactions, the steady-state concentrations for the peroxynitrite are estimated to be in the nanomolar concentration range and can be sustained for long periods of time. Hence, under certain conditions, exposure to peroxynitrite can be significant owing to both the level and duration of exposure. Furthermore, despite the relatively short half-life of peroxynitrite at physiological pH (˜10 ms), its ability to cross cell membranes implies that peroxynitrite generated from a cellular source could influence surrounding target cells within one or two cells diameters. Considering that biological systems exposed to peroxynitrite experience or manifest a multitude of biological effects, including adverse effects on cell viability and function, the degree of potential damage is exacerbated (Szabo et al., Peroxynitrite; biochemistry, pathophysiology and development of therapeutics, Nature Reviews, 6:662-680, 2007).
Reactive Oxygen Species (ROS)
Though there are a number of mechanisms of UV damage in the skin, none is more significant than that due to the action of reactive oxygen species (ROS), also known as free radicals. ROS are generated in increasing quantities with age and are known to damage DNA in general, most especially mitochondrial DNA, as well as cells and tissues. While there is still much to learn, some of the molecular mechanisms upstream of ROS formation have been identified recently with photosensitization by endogenous skin chromophores having emerged as a mechanism linking initial photon absorption with ROS formation in skin (G T Wondarak et al, Identification of Quenchers of Photoexcited States as Novel Agents for Skin Photoprotection, Pharmacol and Experimental Therapeutics, 312(2):482-491, 2005; & references cited therein). Many skin chromophores, including urocanic acid, riboflavin, B6 vitamins, melanin precursors, and porphyrins, are suspected endogenous photosensitizers. Extracellular matrix proteins such as collagen and elastin, which are present in large amounts in the skin and are rich in advanced glycation end products (AGEs) and other cross-link fluorophores, have been identified as potent UVA sensitizers of phot oxidative stress (G T Wondrak et al., Photosensitization of DNA damage by glycated proteins. Photochem Photobiol Sci 1:355-363, 2002). After initial photon absorption, excited singlet states can either relax to the ground state with or without light emission (fluorescence) or undergo intersystem crossing (ISC) with formation of highly reactive biradical triplet states. Photoexcited states exert skin photodamage by direct reaction with substrate molecules, including DNA bases (type I photosensitization) and molecular oxygen (type II photosensitization), leading to ROS formation. Singlet oxygen (1O2), an electronically excited, highly reactive form of molecular oxygen, is formed after energy transfer between the triplet photoexcited state of the sensitizer and ground state triplet oxygen. 1O2 is a widely accepted example of an excited state mediator of skin photodamage involved in UVA-induced mutagenesis, stress signaling, apoptosis, and remodeling of extracellular matrix components during skin photoaging and carcinogenesis (L O Klotz, K O Kroncke, and H Sies, Singlet oxygen-induced signaling effects in mammalian cells, Photochem Photobiol Sci 2: 88-94, 2003).
Paradoxically, some sunscreens act as potent triplet state UV sensitizers, enhancing light-driven formation of ROS and skin cell photodamage (M Gulston and J Knowland, Illumination of human keratinocytes in the presence of the sunscreen ingredient padimate-O and through an SPF-15 sunscreen reduces direct photodamage to DNA but increases strand breaks, Mutat Res 444: 49-60, 1999). Although moderate skin photo-protection has been demonstrated in many experiments on animal and human skin through topical application of antioxidants (L Packer L and G Valacchi G, Antioxidants and the response of skin to oxidative stress: vitamin E as a key indicator. Skin Pharmacol Appl Skin Physiol 15: 282-290, 2002), the therapeutic effectiveness of skin administration of antioxidants is limited by their sacrificial depletion, their pronounced spontaneous redox chemistry, and their negative interference with the highly regulated skin antioxidant network (A Meves A, S N Stock, A Beyerle, M R Pittelkow, and D Peus, Vitamin C derivative ascorbyl palmitate promotes ultraviolet-B-induced lipid peroxidation and cytotoxicity in keratinocytes. J Investig Dermatol 119: 1103-1108, 2002). Harmful interaction of chemical antioxidants with essential redox signaling in human skin may be anticipated because recent reports point to a significant potential for antioxidant enhanced carcinogenesis in transgenic mice with up-regulated antioxidant responses (Y P Lu, Y R Lou, P Yen, H L Newmark, O I Mirochnitchenko, M Inouye, and M T Huang, Enhanced skin carcinogenesis in transgenic mice with high expression of glutathione peroxidase or both glutathione peroxidase and superoxide dismutase. Cancer Res 57: 1468-1474, 1997).
In light of the foregoing, the role of photoexcited states in skin photodamage and carcinogenesis suggests that direct molecular antagonism of photooxidative stress by physical Quencher of Photo-excited States (QPES) compounds has the potential to reduce skin photocarcinogenesis and photoaging. According to the accepted importance of UVA irradiation in sensitized skin photodamage, QPES are predicted to be most efficacious against photodamage caused by UVA irradiation and are therefore intended for combinatorial use with existing agents for skin photoprotection, especially antioxidants and UV sunscreen actives. Thus, QPES could be a functionally synergistic additive in existing sunscreen formulas and may provide other beneficial effects such as enhancement of photostability of sunscreens (E Chatelain and B Gabard, Photostabilization of butyl methoxydibenzoylmethane (Avobenzone) and ethylhexyl methoxycinnamate by bis-ethylhexyloxyphenol methoxyphenyl triazine (Tinsorb S), a new UV broadband filter, Photochem Photobiol 74: 401-406, 2001).
Mitochondrial DNA
The mitochondrion is a tiny structure inside a cell and is the primary generator of energy, in the form of adenosine tri-phosphate (ATP). Mitochondria have their own DNA which determines all of their functions. Mitochondrial DNA (mtDNA) is made up of 16569 base pairs that, when completely intact, make energy for the body; however, subtle changes in the mtDNA, especially that arising from oxidative damage, have dramatic adverse effects on mitochondrial function and energy production as well as longevity. In following, the mitochondrial clock theory of aging is based upon the progressive accumulation of this oxidative damage, especially that damage arising from reactive oxygen species (ROS).
Over the past decade, researchers have identified and measured various forms of endogenous and environmental mitochondrial DNA (mtDNA) damage and have elucidated mtDNA repair pathways. Interestingly, mitochondria do not appear to possess the full range of DNA repair mechanisms that operate in the nucleus: although mtDNA contains the same types of damage that are targets of each nuclear DNA repair pathway, The reduced repair capacity may, in part, explain the high mutation frequency of the mitochondrial chromosome [S D Cline, Mitochondrial DNA Damage and its Consequences for Mitochondrial Gene Expression, Biochim Biophys Acta, 1819(9-10): 979-991, 2012].
More recently, research around the world has identified a specific deletion (or elimination) in mitochondrial DNA that is known to occur in response to aging and UV exposure [S D Cline, Mitochondrial DNA Damage and its Consequences for Mitochondrial Gene Expression, Biochim Biophys Acta, 1819(9-10): 979-991, 2012; references cited therein]. This deletion is called the common aging deletion or the 4977 base pair deletion; though, it is to be appreciated that there are many other mtDNA deletions that occur in response to aging, such as the 520 base pair deletion, etc. Indeed, it has been found that even minor amounts of this deletion severely alter energy production and cellular function.
Clearly mtDNA is susceptible to endogenous and environmental damage and, unlike other DNA in the human body, lacks the full cohort of nuclear DNA repair mechanisms. Consequently, persistent mtDNA damage poses a threat to mitochondrial gene expression, especially mitochondrial polymerase, whose disruption is believed to underlie much, if not most, skin damage and, consequently, many human diseases.
UV Exposure and Formation of Superoxide via NADPH Oxidase
Nicotinamide adenine dinucleotide phosphate oxidase (NADPH) represents the first step that controls the oxidative stress cascade. Strong evidence suggests that NADPH Oxidase or NOX enzymes are major contributors to oxidative damage in pathologic conditions (V Jaquet et al, Small-Molecule NOX Inhibitors: ROS-Generating NADPH Oxidases as Therapeutic Targets, Antioxidants & Redox. Signaling, 11(10): 2535-2552, 2009—Review). In following, it has been established that keratinocytes and fibroblasts generate ROS in response to UV light (S M Beak et al, Biochimie, 86: 425-429, 2004). These ROS responses can be blocked by NADPH oxidase inhibitors, raising the possibility that ROS generation even in response to ultraviolet light is not simply a physicochemical process, but involve NOX enzymes. Furthermore, it has been established that NOX-derived ROS are involved in the regulation of expression and/or activation of matrix metalloproteases (K Bedard & K H Krause, Physiol and Pathophysiol, Physiol Rev 87: 245-313, 2007). While NOX2 occurs in normal cells, both NOX2 and NOX4 are expressed in melanoma cells. On the basis of antisense experiments, NOX4 has been suggested to promote cell growth in melanoma cells (S S Brar et al, Am J Physiol Cell Physiol, 282: c1212-c1224, 2002). Hence, blocking the undesirable actions of NOX enzymes may be a therapeutic strategy for treating oxidative stress-related pathologies, especially those arising from photo-damage.
Additional Factors
Certainly, UV radiation has a marked adverse effect on a number of biomolecular processes in the skin; however, among other factors and influences, air pollution also has a marked adverse effect (P Puri et al., Effects of air pollution on the skin: A review, Indian J Dermatol Venereol Leprol. 2017 83(4):415-423, 2017). Additionally, it is to be appreciated that such skin damage is not limited to the aforementioned biomolecular processes. For example, ultraviolet (UV) light enhances synthesis of matrix metalloproteases (MMPs) in human skin in vivo, and MMP mediated collagen destruction occurs in photo-aging. UV light and air pollutants also induce oxidative stress and pro-inflammatory mediators, such as transcription factors and chemokines, causing inflammation-induced skin damage. Hence, strategies to counter the effects of skin aging due to UV and air pollution exposure must be multifaceted and multidirectional if they are to be effective. Accordingly, efforts to slow down the aging process must endeavor to (1) achieve or establish antioxidant protection to limit direct oxidative damage to the cells, proteins, and DNA, (2) reduce the activity of oxidase enzymes, (3) control or mitigate inflammation and inflammatory responses to minimize inflammation-induced aging, (4) prevent degradation of extracellular matrix proteins by inhibiting matrix metalloproteases (MMPs) and (5) prevent photon- and enzyme-induced skin damage, among others.
Skin Structures
From the foregoing, it is evident that UV radiation has a marked detrimental effect on a molecular level in skin cells, from damage to DNA to the formation of ROS, and that the avenues of damage are numerous and far reaching. Furthermore, damage from exposure to UV radiation is not limited to the molecular level, but affects a host of other structures and processes that adversely affect the skin. However, these adverse effects are not limited to UV radiation exposure. Indeed, chronological aging and diseases, directly and indirectly, have a marked adverse effect on a broad number of biological processes in the skin as well as on the integrity of a number of skin structures and their efficacy and/or operation or function.
Perhaps the key underlying physiological change in aging skin is a thinning and general degradation of the skin, most notably a degradation and/or loss of various cells and/or chemical constituents necessary for maintaining the physiological characteristics of youthful skin. Specifically, as the skin naturally ages, the division rate of skin cells slows down causing an overall reduction in the number of cells and blood vessels that supply nutrients and other necessary building blocks for the skin which results in a significant decrease in the thickness of the epidermis. Concurrently, as the skin ages proteins, especially collagen and elastin fibers in the underlying layers of skin which provide the scaffolding for the surface layers, begin to weaken and deteriorate and/or manifest a deterioration in their cross-linking capabilities causing the skin to lose elasticity as well as resulting in a flattening of and concurrent loss of mechanical properties, including strength and flexibility, particularly, but not exclusively, in the dermal-epidermal junction (Neerken S, Lucassen G W, Bischop M A, Lenderink E, Nuijs T A, J Biomed Opt, 2004 March-April: 9(2)274-81 and Oikarinen, “The Aging of Skin; Chronoaging Versus Photoaging,” Photodermatol. Photoimmunol. Photomed., vol. 7, pp. 3-4. 1990, both of which are incorporated by reference herein in their entirety).
The dermal-epidermal junction (DEJ) is a critical component of the skin and is composed of a network of structural proteins that provide a firm connection between the basal keratinocytes of the epidermis and the dermis. This structural network is made up of (1) the hemidesmosome-anchoring filament complex; (2) the basement membrane comprising two layers, the lamina lucida and the lamin dense, and (3) anchoring fibrils. The lamina lucida is a thinner layer and lies directly beneath the epidermal layer of basal keratinocytes. The thicker lamina dense is in direct contact with the underlying dermis. The basal keratinocytes are connected to the basement membrane via the hemidesmosome-anchoring complex and the basement membrane, in turn, is connected to the dermis via the anchoring fibrils. Each of these components of the DEJ has specific constituents, most notably laminins, integrins tenascin, and above all collagens, specifically collagen IV, and a very precise role to play (Allen J., Br. J. Dermatol. 1997 December; 137 (6): 907-15), (M. Aumailley, Kidney Internat., Vol 47, Suppl. 49 (1995), pp S-4-S-7). Concurrently, these structures are the target of immunologic injury in bullous pemphigoid and epidermolysis bullosa.
Collagen fibers are major elements of the dermis and collagens and the most abundant protein in the human body; the dermis alone is composed of approximately 75% collagen proteins in dry weight. So far, twenty-eight collagen species have been identified. Of these it has been reported that skin contains collagen types 1, 3, 4-7, 13, and 14, with the major collagen in the dermis being collagen type 1. Collagens that associate with the collagen type 1 fiber are classified as FACIT collagens and can provide additional mechanical properties to tissues. Collagens are characterized by repeated glycine-X-Y sequences and form triple-helical structures that are extensively modified after their secretion into the extracellular space. In immature tissues, such as those found in wound healing and fibrosis, collagen type 3 is expressed; however, it is not yet strong enough to support mature connective tissues. As the wound matures, collagen type 1 becomes dominant. Heterotypic type 1 and type 3 collagen fibrils are present in the dermis. Collagen type 4 individually forms a unique filament called a microfilament (T Nemoto et al., Viscoelastic Properties of the Human Dermis and Other Connective Tissues and Its Relevance to Tissue Aging and Aging-Related Disease, Intech, Chapter 7, DOI: 10.5772/50146 and refs cited therein),
Elastic fiber comprises elastin and microfibrils. Since the dermis has be stretched to adapt to the movement of body parts, elasticity is a critical property of the dermis. Elastin, a unique molecule that stretches and shrinks, is secreted as tropoelastin (the soluble precursor of mature elastin) and is subsequently processed and cross-linked within the extracellular space. Cross-linking by lysyl oxidase and desmosine formation is a crucial step for the stabilization of elastin within tissues. Another element in elastic fibers is fibrillin-microfibril. Microfibrils are fibrous elements that are 10 nm in width and are comprised mainly of fibrillins. Fibrillin is a large glycoprotein that is rich in cysteine residues and homotypically assembles into a microfibril in a well-regulated manner. Fibrillins align in a parallel manner, from head to tail, in a staggered fashion within extracellular microfibrils. Other extracellular matrix (ECM) molecules, including microfibril-associated glycoproteins (MAGPs), latent TGF-beta binding proteins (LTBPs), collagen type 16, emilin, and versican, can associate with microfibrils through their binding affinity with fibrillins. Fibulins are yet another elastic fiber component, which can bridge elastin and microfibrils by their binding properties.
The dermis changes prominently with age; for example, the thickness of the dermis becomes thin and wrinkles appear. Biochemical collagen content and histological density of collagen fiber is reduced. Versican is a key molecule for viscoelasticity of the dermis (T Nemoto et al., Viscoelastic Properties of the Human Dermis and Other Connective Tissues and Its Relevance to Tissue Aging and Aging-Related Disease, Intech, Chapter 7, DOI: 10.5772/50146; & references cited therein). Loss or reduction of versican leads to impaired viscoelasticity of the dermis. Versican is heavily accumulated within solar elastosis, which is a hallmark of photo-aged skin and where elastic fiber components, including elastin and fibrillin-1, have accumulated (E F Bernstein et al., Differential Expression of the Versican and Decorin Genes in Photoaged and Sun-Protected Skin. Comparison by Immunohistochemical and Northern Analyses, Lab Invest, 72662669, 1995). Clinically, photo-aged skin is not viscoelastic and shows deep wrinkles
Fibulins are a family of calcium-binding extracellular glycoproteins associated with basement membranes and elastic fibers in vertebrates. The fibulins do not form large homotypic aggregates, in contrast to many other ECM proteins, but they have the ability to join other supramolecular structures as diverse as basement-membrane networks, elastic fibers, several types of microfibrils and proteoglycans aggregates. Fibulin-1 is a prominent component of skin which is essential for the morphology of endothelial cells lining capillary walls and the integrity of small blood vessels [W S Argraves et al., Fibulins: physiological and disease perspectives. EMBO Rep, 4:1127-1131, 2003; R Timpl et al., Fibulins: A versatile family of extracellular matrix proteins, Mol Cell Biol, 4:479-489, 2003 in Viscoelasticity—From Theory to Biological Applications, book edited by Juan de Vicente, ISBN 978-953-51-0841-2, Published: Nov. 7, 2012].
Numerous efforts have been undertaken for improving the dermal-epidermal junction resulting in a number of successful, at least to some extent, techniques. For example, Marionnet et. al. have shown the utility of vitamin C in improving the DEJ formation in an in vitro human reconstructed skin model leading to a DEJ structure closer to that of normal young looking human skin (Marionnet C, Vioux-Chagnoleau C. Pierrard C, Sok J, Asselineau O, Bernard F. Meeting abstracts, 34.sup.th Annual European Society for dermatological Research Meeting, September 2004, Vienna, Austria). Similarly, Fisher et. al. have shown an improvement in the DEJ formation and extracellular matrix proteins arising from retinoids (Fischer G J and Voorhees J, J. Molecular mechanisms of retinoid actions in skin. FASEB J. 10, 1002-1013 (1998).
Certainly, while much effort has been directed to the DEJ, the DEJ is certainly not the only area of focus. Indeed, a number of investigators have shown the beneficial impact of topical application of retinoids on skin appearance as well as on various histological parameters such as a thickening of the epidermis including the stratum granulosum, an increase in the height of epidermal ridges or rates of the DEJ and the number of dermal papillae, a gradual displacement of age-related deposition of dermal elastin by collagen and peptidoaminoglycans, normalization of melanocyte function and an increase in the number of dermal fibroblasts. See, for example, Kligman, U.S. Pat. Nos. 4,603,146 and 4,877,805; Zelickson, A. S., J. Cutaneous Aging Cosmet. Dermatol., 1:4147 (1988); Weiss, J. S., JAMA, 259:527-532 (1988); J. Bhawan, Arch. Dermatol., 127:666-672 (1991); and Kligman, L. H., Connect. Tissue Res., 12:139-150 (1984). Similarly, Varani et. al. have shown vitamin A as antagonizing decreased cell growth and elevated collagen-degrading matrix metalloproteases while concurrently stimulating collagen accumulation in naturally aged human skin, Varani J, et. al., J Investigative Dermatology, 114:480-486, 2000). Dyer et. al. (U.S. Pat. No. 7,351,745) teach a method of applying a physiologically effective amount of an active agent, wherein said active agent is S-Methyl-L-Cysteine and S-Phenyl-L-Cysteine in a dermatologically pharmaceutically or physiologically acceptable vehicle, sufficient to increase expression levels of at least one gene selected from the group consisting of: Beta-catenin, Collagen 4. Collagen 7, Frizzled 10, Estrogen Receptor alpha, Hyaluronic acid synthase, and combinations thereof and for improving the condition and appearance of skin. Bernerd (US Patent Application: 2004/0005342) teaches the use of ascorbic acid or an analogue thereof in a pharmaceutically or cosmetically acceptable medium to increase the synthesis of tenascin and/or collagen VII for reinforcing the cohesion at the DEJ. Dal Farra et. al. (U.S. Pat. No. 7,211,269) teach a method for preparing cosmetic or dermatological compositions of a sufficient amount of peptides of sequence (Gly-Pro-Gln)n-NH2, wherein n is from 1 to 3, and wherein the amino acids can be in the form L, D or DL: these compositions being designed to promote adhesion between skin cells, to enhance cell adhesion, to provide curative and/or preventive treatment for aging skin symptoms (of physiological or solar origin) and to enhance skin appearance. In a preferred embodiment, the peptide is of sequence (Gly-Pro-Gln)2-NH2. Bonte et. al. (U.S. Pat. No. 6,641,848) teach the use of saponins or sapogenols, particularly those extracted from plants such as soya or Medicago, in cosmetology and for the manufacture of pharmaceutical compositions for treating the skin in order to increase the amount of collagen IV in the dermal-epidermal junction. Paufique (U.S. Pat. No. 6,531,132) describes a process for extracting an active principle from yeast whereby the active principle is used to retard the degradation of the dermal-epidermal junction to improve the surface condition of the skin. Dumas et. al. (U.S. Pat. No. 6,495,147) describe the use of D-xylose, esters thereof or oligosaccharides containing D-xylose for stimulating the synthesis and/or secretion of proteoglycans and/or glycosaminoglycans by the keratinocytes of a human in need thereof. Bonte et. al. (U.S. Pat. No. 6,471,972) teach a cosmetic treatment method for fighting against skin aging effects wherein the method comprises the application of at least one agent for promoting the adhesion of the keratinocytes of the epidermal basal layer to the dermal-epidermal junction, especially to the collagen IV of said junction, such as, in particular, a divalent metal salt or complex, preferably magnesium aspartate or magnesium chloride, optionally in association with a stimulant of collagen IV synthesis and/or a stimulant of collagen synthesis. LeSquer et. al. (WO 2002/015869) described combinations of ursolic acid and/or oleanolic acid with a specific palmitoyl pentapeptide Lys-Thr-Thr-Lys-Ser as synergistically increasing/stimulating the neosynthesis of compounds of the DEJ including collagen IV.
Despite all the efforts that have been undertaken to formulate effective compositions for improving the dermal-epidermal junction, current products are not entirely effective, Vitamin C and some of its derivatives are not photochemically or hydrolytically stable. In certain environments, especially in the presence of iron and hydrogen peroxide, Vitamin C can act as a pro-oxidant. Retinoids are very effective, but they also suffer from stability problems. Additionally, retinoids can also cause skin irritation, sensitization and are teratogenic. Plant extracts, if not standardized against key actives, oftentimes are not effective, Peptides are effective, but not fully characterized as yet. For example, though not manifest in short term use, some minor peptide impurities may cause adverse effects over long-term use. Consequently, the user oftentimes finds themselves with no results or an undesired result, e.g., irritation, sensitization, burning sensation, erythema, etc. of the skin.
Alternative approaches to improving the condition or appearance of aging skin that have received increasing attention involve the modulation of extracellular matrix proteins and matrix degrading enzymes and transcription factors. Representative disclosures in this area include: Mancini A, Di Battista J A, “Transcriptional regulation of matrix metalloprotease gene expression in health and disease”, Front Biosci, 11:423-446, 2006. S Reitamo, A Remitz, K Tamai, and J Uitto, “Interleukin-10 modulates type I collagen and matrix metalloprotease gene expression in cultured human skin fibroblasts”, Cin Invest, 1994, 94(6):2489-2492, 1994 von Marcschall Z, Riecken E O, Rosewicz S, “Induction of matrix metalloprotease-1 gene expression by retinoic acid in the human pancreatic tumor cell line Dan-G”, Br J Cancer, 80(7):935-939, 1999. Bair E L, Massey C P, Tran N L, Borchers A H, Heimark R L, Cress A E, Bowden G T, “Integrin- and cadherin-mediated induction of the matrix metalloprotease matrilysin in co-cultures of malignant oral squamous cell carcinoma cells and dermal fibroblasts”, Exp Cell Res, 270(2):259-267, 2001. Nagahara 5, Matsuda 1, “Cell-substrate and cell-cell interactions differently regulate cytoskeletal and extracellular matrix protein gene expression”, J Biomed Mater Res, 32(4):677-86, 1996 Smits P. Poumay Y, Karperien M, Tylzanowski P, Wauters J, Huylebroeck D, Ponec M and Merregaert J. “Differentiation-Dependent Alternative Splicing and Expression of the Extracellular Matrix Protein 1 Gene in Human Keratinocytes”, J Invest Dermatol, 114:718-724, 2000, Reunamen N, Westermarck J, Hakkinen L, Holmstrom, Elo I, Eriksson J E, Kahari V M, “Enhancement of fibroblast collagenase (matrix metalloproteinase-1) gene expression by ceramide is mediated by extracellular signal-regulated and stress-activated protein kinase pathways”, J Biol Chem, 273(9):5137-45, 1998. McKay I A, Winyard P, Leigh I M, Bustin S A, “Nuclear transcription factors: potential targets for new modes of intervention in skin disease”, Br J Dermatol, 131(5):591-597, 1994.
As evident from the foregoing discussion, skin aging is a complex biological process influenced by a combination of endogenous or intrinsic and exogenous or extrinsic factors. Because of the fact that skin health and beauty is considered one of the principal factors representing overall “well-being” and the perception of “health” in humans, the development of anti-aging strategies has long been and continues to be a key focus of research and development efforts in the health and beauty arena [R Ganceviciene et al., Skin anti-aging strategies, Dermatoendocrinol. 4(3): 308-319, 2012 and refs cited therein], Chronic photodamage of the skin manifests itself as extrinsic skin aging (photoaging) wherein DNA photodamage and UV-generated reactive oxygen species (ROS) are the initial molecular events that lead to most of the typical histological and clinical manifestations of chronic photodamage of the skin. Wrinkling and pigmentary changes are directly associated with premature photo-aging and are considered its most important cutaneous manifestations. To date research and development efforts have focused on two main groups of compounds for use as anti-aging agents: the antioxidants and the cell regulators. Antioxidants, such as vitamins, polyphenols and flavonoids, reduce collagen degradation by reducing the concentration of free radicals in the tissues. Cell regulators, such as retinols, peptides and growth factors (GF), have direct effects on collagen metabolism and influence collagen production, Unfortunately, these efforts have been limited, providing marginal results owing to specificity of their actions and the myriad of mechanisms and processes involved, as detailed above.
Accordingly, there is an urgent and huge unmet need for effective methods and compositions that can prevent skin damage and reverse photoaging and/or chronological aging on multiple fronts simultaneously, thereby preventing and/or mitigating and/or delaying its onset.
Specifically, there is a need to provide methods and compositions having significant and marked efficacy in minimizing and/or preventing, most especially in reversing, chronic UV and air pollutant-induced skin damage on the molecular, including DNA, level as well as through the reduction in other UV-induced related biomarkers.